We have proposed an antiviral strategy against the human immunodeficiency virus (HIV-1) which employs a defective interfering HIV-1 particle that can be targeted to HIV-1 infected cells. Expression of this defective virus would interfere with HIV-1 replication and, at the same time, use the structural and regulatory proteins of HIV-1 for its own replication and spread throughout the population of HIV-1-infected cells. Toward the generation of such defective interfering HIV-1 particles, we have isolated a stable cell line which encodes a complete copy of one of our recombinant defective interfering HIV-1 proviruses. This provirus encodes a chimeric CD4/Env protein as well as a multitarget-ribozyme under control of the HIV-1 LTR. These cells could be infected by HIV-1 as efficiently as cells without the defective provirus. This was shown by HIV-1 proviral integration at 10 hr postinfection. Challenge of the cells by HIV-1 infection resulted in a transactivation of gene expression from the LTR of the defective provirus. As compared to the cell line which does not contain the defective interfering provirus, the amount of spliced and unspliced mRNAs from the superinfecting wild type virus was drastically reduced. The amount of infectious virus released from these cells was reduced up to 300 fold, as were syncytia formation and the amounts of p24 antigen in cell supernatants. The infectivity of the released virus was also reduced 2- to 4-fold as a result of a change in the make-up of the released virus, which seemed to contain less viral envelope protein. Polyadenylated defective interfering HIV-1 RNA was packaged into virus particles, which opens the possibility for a transfer of the interfering genes through HIV-1 like particles in the future. Challenge of the same cells with HIV-2 only resulted in an approximately 2-fold reduction in the virus yield. We conclude that the main mechanism of HIV-1 inhibition in this cell line was caused by the multitarget- ribozymes encoded by the defective provirus and the resultant sequence- specific degradation of HIV-1 transcripts. These results demonstrate that inducible, defective interfering HIV-1 proviruses can be very efficient in an intracellular immunization against HIV-1.